Pulse-sampling fluorescence lifetime imaging: evaluation of photon economy.

This Letter presents an experimental study comparing the photon rate and photon economy of pulse sampling fluorescence lifetime imaging (PS-FLIm) with the conventional time-correlated single photon counting (TCSPC) technique. We found that PS-FLIm has a significantly higher photon detection rate (200 MHz) compared with TCSPC (2-8 MHz) but lower photon economy (4-5 versus 1-1.3). The main factor contributing to the lower photon economy in PS-FLIm is laser pulse variability. These results demonstrate that PS-FLIm offers 25× faster imaging speed than TCSPC while maintaining room light rejection in clinical settings. This makes PS-FLIm a robust technique for clinical applications.

Engineering the gain and bandwidth in avalanche photodetectors.

Avalanche and Single-Photon Avalanche photodetectors (APDs and SPADs) rely on the probability of photogenerated carriers to trigger a multiplication process. Photon penetration depth plays a vital role in this process. In silicon APDs, a significant fraction of the short visible wavelengths is absorbed close to the device surface that is typically highly doped to serve as a contact. Most of the photogenerated carriers in this region can be lost by recombination, get slowly transported by diffusion, or multiplied with high excess noise. On the other hand, the extended penetration depth of near-infrared wavelengths requires thick semiconductors for efficient absorption. This diminishes the speed of the devices due to the long transit time in the thick absorption layer that is required for detecting most of these photons. Here, we demonstrate that it is possible to drive photons to a critical depth in a semiconductor film to maximize their gain-bandwidth performance and increase the absorption efficiency. This approach to engineering the penetration depth for different wavelengths in silicon is enabled by integrating photon-trapping nanoholes on the device surface. The penetration depth of short wavelengths such as 450 nm is increased from 0.25 µm to more than 0.62 µm. On the other hand, for a long-wavelength like 850 nm, the penetration depth is reduced from 18.3 µm to only 2.3 µm, decreasing the device transit time considerably. Such capabilities allow increasing the gain in APDs by almost 400× at 450 nm and by almost 9× at 850 nm. This engineering of the penetration depth in APDs would enable device designs requiring higher gain-bandwidth in emerging technologies such as Fluorescence Lifetime Microscopy (FLIM), Time-of-Flight Positron Emission Tomography (TOF-PET), quantum communications systems, and 3D imaging systems.

Assessment of Murine Colon Inflammation Using Intraluminal Fluorescence Lifetime Imaging.

Inflammatory bowel disease (IBD) is typically diagnosed by exclusion years after its onset. Current diagnostic methods are indirect, destructive, or target overt disease. Screening strategies that can detect low-grade inflammation in the colon would improve patient prognosis and alleviate associated healthcare costs. Here, we test the feasibility of fluorescence lifetime imaging (FLIm) to detect inflammation from thick tissue in a non-destructive and label-free approach based on tissue autofluorescence. A pulse sampling FLIm instrument with 355 nm excitation was coupled to a rotating side-viewing endoscopic probe for high speed (10 mm/s) intraluminal imaging of the entire mucosal surface (50-80 mm) of freshly excised mice colons. Current results demonstrate that tissue autofluorescence lifetime was sensitive to the colon anatomy and the colonocyte layer. Moreover, mice under DSS-induced colitis and 5-ASA treatments showed changes in lifetime values that were qualitatively related to inflammatory markers consistent with alterations in epithelial bioenergetics (switch between ß-oxidation and aerobic glycolysis) and physical structure (colon length). This study demonstrates the ability of intraluminal FLIm to image mucosal lifetime changes in response to inflammatory treatments and supports the development of FLIm as an in vivo imaging technique for monitoring the onset, progression, and treatment of inflammatory diseases.

Multimodal Scanning Microscope Combining Optical Coherence Tomography, Raman Spectroscopy and Fluorescence Lifetime Microscopy for Mesoscale Label-Free Imaging of Tissue.

Multimodal optical imaging of tissue has significant potential to become an indispensable diagnostic tool in clinical pathology. Conventional bright-field microscopy provides contrast based on attenuation or reflectance of light, having no depth-related information and no molecular specificity. Recent developments in biomedical optics have introduced a variety of optical modalities, such as Raman spectroscopy (RS), fluorescence lifetime imaging microscopy (FLIM) of endogenous fluorophores, optical coherence tomography (OCT), and others, which provide a distinct characteristic, i.e., molecular, chemical, and morphological information, of the sample. To harvest the full analytical potential of those modalities, we have developed a novel multimodal imaging system, which allows the co-registered acquisition of OCT/FLIM/RS on a single device. The present implementation allows the investigation of biological tissues in the mesoscale range, 0.1-5 mm in a correlated manner. Due to the co-registered acquisition of the modalities, it is possible to directly compare and evaluate the corresponding information between the three modalities. Moreover, by additionally preparing and characterizing entire pathological hematoxylin and eosin (H&E) slides of head and neck biopsies, it is also possible to correlate the multimodal spectroscopic information to any location of the ground truth H&E information. To the best of our knowledge, this is the first development and implementation of a compact and clinically applicable multimodal scanning microscope, which combines OCT, FLIM, and RS together with the possibility for co-registering H&E information for a morpho-chemical tissue characterization and a correlation with the pathological ground truth (H&E) of the underlying signal origin directly in a clinical environment.

Multispectral fluorescence lifetime imaging device with a silicon avalanche photodetector.

We report the design, development, and characterization of a novel multi-spectral fluorescence lifetime measurement device incorporating solid-state detectors and automated gain control. For every excitation pulse (∼1 µJ, 600 ps), this device records complete fluorescence decay from multiple spectral channels simultaneously within microseconds, using a dedicated UV enhanced avalanche photodetector and analog to digital convert (2.5 GS/s) in each channel. Fast (<2 ms) channel-wise dynamic range adjustment maximizes the signal-to-noise ratio. Fluorophores with known lifetime ranging from 0.5-6.0 ns were used to demonstrate the device accuracy. Current results show the clear benefits of this device compared to existing devices employing microchannel-plate photomultiplier tubes. This is demonstrated by 5-fold reduction of lifetime measurement variability in identical conditions, independent gain adjustment in each spectral band, and 4-times faster imaging speed. The use of solid-state detectors will also facilitate future improved performance and miniaturization of the instrument.

Intraoperative Mapping of Parathyroid Glands Using Fluorescence Lifetime Imaging.

BACKGROUND: Hypoparathyroidism is a common complication following thyroidectomy. There is a need for technology to aid surgeons in identifying the parathyroid glands. In contrast to near infrared technologies, fluorescence lifetime imaging (FLIm) is not affected by ambient light and may be valuable in identifying parathyroid tissue, but has never been evaluated in this capacity. METHODS: We used FLIm to measure the UV induced (355 nm) time-resolved autofluorescence signatures (average lifetimes in 3 spectral emission channels) of thyroid, parathyroid, lymphoid and adipose tissue in 21 patients undergoing thyroid and parathyroid surgery. The Mann-Whitney U test was used to assess the ability of FLIm to discriminate normocellular parathyroid from each of the other tissues. Various machine learning classifiers (random forests, neural network, support vector machine) were then evaluated to recognize parathyroid through a leave-one-out cross-validation. RESULTS: Statistically significant differences in average lifetime were observed between parathyroid and each of the other tissue types in spectral channels 2 and 3 respectively. The largest change was observed between adipose tissue and parathyroid (P < 0.001), while less pronounced but still significant changes were observed when comparing parathyroid with lymphoid tissue (P < 0.05) and thyroid (P < 0.01). A random forest classifier trained on average lifetimes was found to detect parathyroid tissue with 100% sensitivity and 93% specificity at the acquisition run level. CONCLUSION: We found that FLIm derived parameters can distinguish the parathyroid glands and other adjacent tissue types and has promise in scanning the surgical field to identify parathyroid tissue in real-time.

FLIm-Guided Raman Imaging to Study Cross-Linking and Calcification of Bovine Pericardium.

Bovine pericardium (BP) is a vascular biomaterial used in cardiovascular surgery that is typically cross-linked for masking antigenicity and enhance stability. There is a need for biochemical evaluation of the tissue properties prior to implantation to ensure that quality and reliability standards are met. Here, engineered antigen removed BP (ARBP) that was cross-linked with 0.2% and 0.6% glutaraldehyde (GA), and further calcified in vitro to simulate graft calcifications upon implantation was characterized nondestructively using fluorescence lifetime imaging (FLIm) to identify regions of interest which were then assessed by Raman spectroscopy. We observed that the tissue fluorescence lifetime shortened, and that Raman bands at 856, 935, 1282, and 1682 cm-1 decreased, and at 1032 and 1627 cm-1 increased with increasing GA cross-linking. Independent classification analysis based on fluorescence lifetime and on Raman spectra discriminated between GA-ARBP and untreated ARBP with an accuracy of 91% and 66%, respectively. Pearson's correlation analysis showed a strong correlation between pyridinium cross-links measured with high-performance liquid chromatography and fluorescence lifetime measured at 380-400 nm (R = -0.76, p = 0.00094), as well as Raman bands at 856 cm-1 for hydroxy-proline (R = -0.68, p = 0.0056) and at 1032 cm-1 for hydroxy-pyridinium (R = 0.74, p = 0.0016). Calcified areas of GA cross-linked tissue showed characteristic hydroxyapatite (959 and 1038 cm-1) bands in the Raman spectrum and fluorescence lifetime shortened by 0.4 ns compared to uncalcified regions. FLIm-guided Raman imaging could rapidly identify degrees of cross-linking and detected calcified regions with high chemical specificity, an ability that can be used to monitor tissue engineering processes for applications in regenerative medicine.

FLIm and Raman Spectroscopy for Investigating Biochemical Changes of Bovine Pericardium upon Genipin Cross-Linking.

Biomaterials used in tissue engineering and regenerative medicine applications benefit from longitudinal monitoring in a non-destructive manner. Label-free imaging based on fluorescence lifetime imaging (FLIm) and Raman spectroscopy were used to monitor the degree of genipin (GE) cross-linking of antigen-removed bovine pericardium (ARBP) at three incubation time points (0.5, 1.0, and 2.5 h). Fluorescence lifetime decreased and the emission spectrum redshifted compared to that of uncross-linked ARBP. The Raman signature of GE-ARBP was resonance-enhanced due to the GE cross-linker that generated new Raman bands at 1165, 1326, 1350, 1380, 1402, 1470, 1506, 1535, 1574, 1630, 1728, and 1741 cm-1. These were validated through density functional theory calculations as cross-linker-specific bands. A multivariate multiple regression model was developed to enhance the biochemical specificity of FLIm parameters fluorescence intensity ratio (R2 = 0.92) and lifetime (R2 = 0.94)) with Raman spectral results. FLIm and Raman spectroscopy detected biochemical changes occurring in the collagenous tissue during the cross-linking process that were characterized by the formation of a blue pigment which affected the tissue fluorescence and scattering properties. In conclusion, FLIm parameters and Raman spectroscopy were used to monitor the degree of cross-linking non-destructively.

Broadband, freeform focusing micro-optics for a side-viewing imaging catheter.

Successful implementation of a catheter-based imaging system relies on the integration of high-performance miniaturized distal end optics. Typically, compensation of chromatic dispersion, as well as astigmatism introduced by the device's sheath, can be addressed only by combining multiple optical elements, adversely impacting size and manufacturability. Here, we present a 300×300×800 µm3 monolithic optic that provides high optical performances over an extended wavelength range (near UV-visible-IR) with minimal chromatic aberrations. The design of the optic, fully optimized using standard optical simulation tools, provides the ability to freely determine aperture and working distance. Manufacturing is cost effective and suited for prototyping and production alike. The experimental characterization of the optic demonstrates a good match with simulation results and performances well suited to both optical coherence tomography and fluorescence imaging, thus paving the way for high-performance multimodal endoscopy systems.

Fiber-based platform for synchronous imaging of endogenous and exogenous fluorescence of biological tissue.

Endogenous and exogenous fluorescence emission from biological samples encodes complementary information. Here we report, to the best of our knowledge, the first results from an optical imaging platform with interleaved excitation and detection of exogenous and endogenous fluorescence from tissue samples using a single flexible multimode fiber that delivers the excitation beam and collects the emitted light. A custom-built reflective optical chopper wheel with synchronized rotation temporally multiplexes an autofluorescence lifetime imaging apparatus with an intensity-based fluorescence module tailored to imaging green fluorescent protein. We demonstrate the functionality of such platform imaging dyes of varying fluorescence signatures and resolving cellularized areas on bio-engineered tissue constructs.

Multiscale, multispectral fluorescence lifetime imaging using a double-clad fiber.

Fiber-based imaging of tissue autofluorescence using ultraviolet (UV) excitation is a highly flexible tool used to probe structure and composition. In this Letter, we report, to the best of our knowledge, the first results from a single-fiber imaging system employing a custom double-clad fiber to acquire multispectral fluorescence lifetime images at two distinct spatial resolutions. We characterize the lateral point spread function and fluorescent background of the system and show how enhanced resolution can identify features such as trabeculae in ex vivo murine bone samples.

Hybrid intravascular imaging: recent advances, technical considerations, and current applications in the study of plaque pathophysiology.

Cumulative evidence from histology-based studies demonstrate that the currently available intravascular imaging techniques have fundamental limitations that do not allow complete and detailed evaluation of plaque morphology and pathobiology, limiting the ability to accurately identify high-risk plaques. To overcome these drawbacks, new efforts are developing for data fusion methodologies and the design of hybrid, dual-probe catheters to enable accurate assessment of plaque characteristics, and reliable identification of high-risk lesions. Today several dual-probe catheters have been introduced including combined near infrared spectroscopy-intravascular ultrasound (NIRS-IVUS), that is already commercially available, IVUS-optical coherence tomography (OCT), the OCT-NIRS, the OCT-near infrared fluorescence (NIRF) molecular imaging, IVUS-NIRF, IVUS intravascular photoacoustic imaging and combined fluorescence lifetime-IVUS imaging. These multimodal approaches appear able to overcome limitations of standalone imaging and provide comprehensive visualization of plaque composition and plaque biology. The aim of this review article is to summarize the advances in hybrid intravascular imaging, discuss the technical challenges that should be addressed in order to have a use in the clinical arena, and present the evidence from their first applications aiming to highlight their potential value in the study of atherosclerosis.

Simultaneous, label-free, multispectral fluorescence lifetime imaging and optical coherence tomography using a double-clad fiber.

We present a novel fiber-based imaging platform that allows simultaneous fluorescence lifetime imaging (FLIm) and optical coherence tomography (OCT) using a double-clad fiber. This platform acquires co-registered images showing structural and compositional contrast in unlabeled biological samples by scanning the fiber tip across the sample surface. In this Letter, we report a characterization of each modality and show examples of co-registered FLIm and OCT images acquired from a lemon segment and a section of human coronary artery. The close comparison between the combined FLIm and OCT images and a co-registered histology section provides a qualitative validation of the technique and highlights its potential for minimally invasive, multimodal imaging of tissue structure and composition.

Combined fiber probe for fluorescence lifetime and Raman spectroscopy.

In this contribution we present a dual modality fiber optic probe combining fluorescence lifetime imaging (FLIm) and Raman spectroscopy for in vivo endoscopic applications. The presented multi-spectroscopy probe enables efficient excitation and collection of fluorescence lifetime signals for FLIm in the UV/visible wavelength region, as well as of Raman spectra in the near-IR for simultaneous Raman/FLIm imaging. The probe was characterized in terms of its lateral resolution and distance dependency of the Raman and FLIm signals. In addition, the feasibility of the probe for in vivo FLIm and Raman spectral characterization of tissue was demonstrated. Graphical Abstract An image comparison between FLIm and Raman spectroscopy acquired with the bimodal probe onseveral tissue samples.

Dual modality intravascular catheter system combining pulse-sampling fluorescence lifetime imaging and polarization-sensitive optical coherence tomography.

The clinical management of coronary artery disease and the prevention of acute coronary syndromes require knowledge of the underlying atherosclerotic plaque pathobiology. Hybrid imaging modalities capable of comprehensive assessment of biochemical and morphological plaques features can address this need. Here we report the first implementation of an intravascular catheter system combining fluorescence lifetime imaging (FLIm) with polarization-sensitive optical coherence tomography (PSOCT). This system provides multi-scale assessment of plaque structure and composition via high spatial resolution morphology from OCT, polarimetry-derived tissue microstructure, and biochemical composition from FLIm, without requiring any molecular contrast agent. This result was achieved with a low profile (2.7 Fr) double-clad fiber (DCF) catheter and high speed (100 fps B-scan rate, 40 mm/s pullback speed) console. Use of a DCF and broadband rotary junction required extensive optimization to mitigate the reduction in OCT performance originating from additional reflections and multipath artifacts. This challenge was addressed by the development of a broad-band (UV-visible-IR), high return loss (47 dB) rotary junction. We demonstrate in phantoms, ex vivo swine coronary specimens and in vivo swine heart (percutaneous coronary access) that the FLIm-PSOCT catheter system can simultaneously acquire co-registered FLIm data over four distinct spectral bands (380/20 nm, 400/20 nm, 452/45 nm, 540/45 nm) and PSOCT backscattered intensity, birefringence, and depolarization. The unique ability to collect complementary information from tissue (e.g., morphology, extracellular matrix composition, inflammation) with a device suitable for percutaneous coronary intervention offers new opportunities for cardiovascular research and clinical diagnosis.

Ultrasound backscatter microscopy for imaging of oral carcinoma.

OBJECTIVES: Ultrasound backscatter microscopy (UBM), or ultrasound biomicroscopy, is a noninvasive, label-free, and ionizing radiation-free technique allowing high-resolution 3-dimensional structural imaging. The goal of this study was to evaluate UBM for resolving anatomic features associated with squamous cell carcinoma of the oral cavity. METHODS: The study was conducted in a hamster buccal pouch model. A carcinogen was topically applied to cheeks of 14 golden Syrian hamsters. Six additional hamsters served as healthy controls. A high-frequency (41 MHz, 6-mm focal depth, lateral and axial resolutions of 65 and 37 µm, respectively) UBM system was used for scanning the oral cavity after 14 weeks of carcinogen application. Histologic analyses were conducted on scanned regions. RESULTS: The histologic structure of buccal tissue and microvasculature networks could be visualized from the UBM images. Epithelial and mucosal hypertrophy and neoplastic changes were identified in animals subjected to the carcinogen. In animals with invasive squamous cell carcinoma, lesion development and destruction of the structural integrity of tissue layers were noted. CONCLUSIONS: In this pilot study, UBM generated sufficient contrast for morphologic features associated with oral carcinoma compared to healthy tissue. This modality may present a practical technique for detection of oral neoplasms that is potentially translatable to humans.

Endoscopic fluorescence lifetime imaging for in vivo intraoperative diagnosis of oral carcinoma.

A clinically compatible fluorescence lifetime imaging microscopy (FLIM) system was developed. The system was applied to intraoperative in vivo imaging of head and neck squamous cell carcinoma (HNSCC). The endoscopic FLIM prototype integrates a gated (down to 0.2 ns) intensifier imaging system and a fiber-bundle endoscope (0.5-mm-diameter, 10,000 fibers with a gradient index lens objective 0.5 NA, 4-mm field of view), which provides intraoperative access to the surgical field. Tissue autofluorescence was induced by a pulsed laser (337 nm, 700 ps pulse width) and collected in the 460 ± 25 nm spectral band. FLIM experiments were conducted at 26 anatomic sites in ten patients during head and neck cancer surgery. HNSCC exhibited a weaker florescence intensity (~50% less) when compared with healthy tissue and a shorter average lifetime (τ(HNSCC) = 1.21 ± 0.04 ns) than the surrounding normal tissue (τN = 1.49 ± 0.06 ns). This work demonstrates the potential of FLIM for label-free head and neck tumor demarcation during intraoperative surgical procedures.

Multimodal fluorescence lifetime imaging and optical coherence tomography for longitudinal monitoring of tissue-engineered cartilage maturation in a preclinical implantation model.

Significance: Cartilage tissue engineering is a promising strategy for effective curative therapies for treatment of osteoarthritis. However, tissue engineers depend predominantly on time-consuming, expensive, and destructive techniques as quality control to monitor the maturation of engineered cartilage. This practice can be impractical for large-scale biomanufacturing and prevents spatial and temporal monitoring of tissue growth, which is critical for the fabrication of clinically relevant-sized cartilage constructs. Nondestructive multimodal imaging techniques combining fluorescence lifetime imaging (FLIm) and optical coherence tomography (OCT) hold great potential to address this challenge. Aim: The feasibility of using multimodal FLIm-OCT for nondestructive, spatial, and temporal monitoring of self-assembled cartilage tissue maturation in a preclinical mouse model is investigated. Approach: Self-assembled cartilage constructs were developed for 4 weeks in vitro followed by 4 weeks of in vivo maturation in nude mice. Sterile and nondestructive in situ multispectral FLIm and OCT imaging were carried out at multiple time points ( t = 2 , 4, and 8 weeks) during tissue development. FLIm and 3D volumetric OCT images were reconstructed and used for the analysis of tissue biochemical homogeneity, morphology, and structural integrity. A biochemical homogeneity index was computed to characterize nonhomogeneous tissue growth at different time points. OCT images were validated against histology. Results: FLIm detects heterogenous extracellular matrix (ECM) growth of tissue-engineered cartilage. The outer edge of the tissue construct exhibited longer fluorescence lifetime in 375 to 410 and 450 to 485 nm spectral channels, indicating increase in collagen content. Significant ( p < 0.05 ) decrease of construct homogeneity index was observed between t = 2 weeks and t = 4 weeks. Both FLIm and OCT images revealed defects (voids) at the center of the tissue construct during in vitro culture ( t = 2 and 4 weeks). Cyst formation during in vivo culture was detected by OCT and confirmed with histology. Conclusions: The ability of multimodal FLIm-OCT to nondestructively monitor the heterogenous growth of engineered tissue constructs in situ is demonstrated. Spatial and temporal variation of construct ECM component was detected by FLIm. OCT reveals structural defects (voids and cysts). This multimodal approach has great potential to replace costly destructive tests in the manufacturing of tissue-engineered medical products, facilitating their clinical translation.

Intraoperative detection of IDH-mutant glioma using fluorescence lifetime imaging.

Identifying isocitrate dehydrogenase (IDH)-mutation and glioma subtype during surgery instead of days later can aid in modifying tumor resection strategies for better survival outcomes. We report intraoperative identification of IDH-mutant glioma (N = 12 patients) with a clinically compatible fluorescence lifetime imaging (FLIm) device (excitation: 355 nm; emission spectral bands: 390/40 nm, 470/28 nm, 542/50 nm). The fluorescence-derived parameters were analyzed to study the optical contrast between IDH-mutant tumors and surrounding brain tissue. IDH-mutant oligodendrogliomas exhibited shorter lifetimes (3.3 ± 0.1 ns) than IDH-mutant astrocytomas (4.1 ± 0.1 ns). Both IDH-mutant glioma subtypes had shorter lifetimes than white matter (4.6 ± 0.4 ns) but had comparable lifetimes to cortex. Lifetimes also increased with malignancy grade within IDH-mutant oligodendrogliomas (grade 2: 2.96 ± 0.08 ns, grade 3: 3.4 ± 0.3 ns) but not within IDH-mutant astrocytomas. The current results support the feasibility of FLIm as a surgical adjuvant for identifying IDH-mutant glioma tissue.

In vivo characterization of the human glioblastoma infiltrative edge with label-free intraoperative fluorescence lifetime imaging.

Challenges in identifying a glioblastoma's infiltrative edge during neurosurgical procedures result in rapid recurrence. A label-free fluorescence lifetime imaging (FLIm) device was used to evaluate glioblastoma's infiltrative edge in vivo in 15 patients (89 samples). FLIm data were analyzed according to tumor cell density, infiltrating tissue type (gray and white matter), and diagnosis history (new or recurrent). Infiltrations in white matter from new glioblastomas showed decreasing lifetimes and a spectral red shift with increasing tumor cell density. Areas of high versus low tumor cell density were separated through a linear discriminant analysis with a ROC-AUC=0.74. Current results support the feasibility of intraoperative FLIm for real-time in vivo brain measurements and encourage refinement to predict glioblastoma infiltrative edge, underscoring the ability of FLIm to optimize neurosurgical outcomes.